Study On The Expansion Of Umbilical Cord Blood Mononuclear Cells Supported By Mesenchymal Stem Cells From Umbilical Cord Blood In Combination Of Exogenous Cytokines

Objective To explore a method of isolating and culturing mesenchymal stem cells(MSCs) from umbilical cord blood in vitro and the preliminary identification by cellular surface antigen analys. Moreover, to study the role of mesenchymal stem cells which derived from umbilical cord blood(UCB-MSCs)with exogenous cytokines in supporting the expansion of umbilical cord blood mononuclear cells(UCB-MNCs)in vitro and the optimal harvest time. In order to provide an experimental foundation for hematopoietic stem cell transplantation (HSCT).Methods MSCs were obtained from fresh umbilical cord blood by gradient centrifugation with Hespan and Ficoll-Hypaque, then anchored culture in MesencultTM by adherence screening method. The UCB-MSCs cellular surface antigens, including CD34, CD133, CD29, CD105, CD44, were analyzed on FACScan flow cytometer. Then MNCs separated from fresh umbilical cord blood cultured in a serum-free system -Stem spanTM (with UCB-MSCs as trophoblast cells / exogenous cytokines supported or not) .On the day 0, 7, 10,14 and 18,total MNCs,CD34十/CD133十cells and cellular cycle were quantitated by FACScan flowcytometer, also hematopoietic progenitor cells were assessed by semisolid culture assay.Results The results showed that MSCs which isolated and cultured from umbilical cord blood were uniformly negative for CD34 and CD133, but positive for CD29, CD105 and CD44. Compared with other groups, the expansion fold of total MNCs,CD34十cells,CD133十cells,cellular cycle(stage G2+M+S)proportion and colony forming units(CFU)were highest in the group which had UCB-MSCs as trophoblast cells and exogenous cytokines (P<0.05) at the same time, and the above parameters reached the peak values(P<0.05) at day 10, furthermore this culture system could maintain at least 18 days.Conclusion Mesenchymal stem cells (MSCs) could be isolated and expand-cultured from umbilical cord blood by gradient centrifugation and adherence screening method. The preliminary identification of MSCs phenotype could be accomplished by flowcytometry. UCB-MSCs with exogenous cytokines could efficiently support the expansion of UCB-MNCs in vitro. Days 10-14 may be an optimal harvest time.